Dual bead assays using cleavable spacers and/or ligation to improve specificity and sensitivity including related methods and apparatus

• US 20050069923A1
• Filed: 07/12/2004
• Published: 03/31/2005
• Est. Priority Date: 07/08/1996
• Status: Abandoned Application

• Current Assignee: Brigitte Chau Phan, Jorma Antero Virtanen, Kary Banks Mullis
• Original Assignee: Brigitte Chau Phan, Jorma Antero Virtanen, Kary Banks Mullis
• Inventors: Mullis, Kary Banks, Phan, Brigitte Chau, Virtanen, Jorma Antero
• Application Number:
  US10/889,518
• Publication Number:
  US 20050069923A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

Methods for deceasing non-specific bindings of beads in dual bead assays and related optical bio-discs and disc drive systems. The methods include determining the suitability of a test solid phase for purposes of use in a dual bead assay. The method also includes identifying whether a target agent is present in a biological sample and involves mixing capture beads, reporter beads, and a biological sample. The mixing is performed under binding conditions to permit formation of a dual bead complex if the target agent is present in the sample. The reporter bead and capture bead are each bound to the target agent. Cleavable spacers or displacement linkers may be used in forming the dual bead complexes. The methods also include placing the capture beads and the reporter beads spatially proximally, performing a ligation reaction employing a ligase, and isolating the dual bead complex from the mixture to obtain the isolate. The isolate is exposed to the capture field on a disc and the capture field is having a capture agent that binds to the dual bead complex. The ligation reaction enables covalent binding between capture probe and reporter probe. The ligation also reaction enhances the sensitivity of the dual bead assay.

Claims

1. A method using a detachable linker to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a dual bead complex including at least one reporter bead and at least one capture bead, said capture bead has at least one transport probe and said reporter bead has at least one signal probe, the beads being linked together by a cleavable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

cleaving the cleavable spacers of the dual bead complexes so that only complexes not associated with said the target remain in the dual bead formation;

performing a ligation reaction to introduce a covalent bond between said transport probe and said signal probe to thereby strengthen the bond between the capture bead and the reporter bead;

isolating the remaining dual bead complexes from solution to obtain an isolate;

exposing the isolate to a capture field on an optical bio disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence the dual bead complex in the disc to indicate that the target is present in the sample.

2. A method using a displaceable member to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a dual bead complex including at least one reporter bead and at least one capture bead, wherein said capture bead has at least one transport probe and said reporter bead has at least one signal probe, the beads being linked together by a displaceable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

displacing the displaceable spacers of the dual bead complexes so that only complexes associated with the target remain in the dual bead formation;

performing a ligation reaction to introduce a covalent bond between said transport probe and said signal probe to thereby strengthen the bond between the capture bead and the reporter bead;

isolating the remaining dual bead complexes from solution to obtain an isolate;

exposing the isolate to a capture field on an optical bio disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex in the disc to indicate that the target is present in the sample.

3. A method using a displaceable member to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a dual bead complex including at least one reporter bead and at least one capture bead, the beads being linked together by a displaceable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

displacing the displaceable spacers of the dual bead complexes using a displacement probe so that only complexes associated with the target remain in the dual bead formation;

isolating the remaining dual bead complexes from solution to obtain an isolate;

exposing the isolate to a capture field on an optical bio disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex in the disc to indicate that the target is present in the sample.

4. A method using ligation to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a plurality of capture beads each of having at least one transport probe affixed thereto;

preparing a plurality of reporter beads each having at least one signal probe affixed thereto;

mixing said capture beads, said reporter beads, and a sample to be tested for the presence of a target;

allowing any target present in the sample to bind to the transport and reporter probes thereby forming a dual bead complex including at least one reporter bead and one capture bead; and

performing a ligation reaction to introduce a covalent bond between the transport probes and the reporter probes to thereby strengthen the bond between the capture bead and the reporter bead so that when the dual bead complexes are processed in a fluidic circuit of a rotating optical bio-disc, said strengthened bond withstands any rotational forces acting thereon.

5. The method according to claim 16 including the further steps of:

isolating the dual bead complex from solution to obtain the isolate;

exposing the isolate to a capture field on said optical bio-disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex in the disc to indicate that the target agent is present in the sample.

6. The method according to claim 16 wherein said mixing, allowing, and performing steps are carried out in said optical bio-disc.

7. The method according to claim 17 wherein said isolating, exposing, and detecting steps are performed in association with said optical bio-disc.

8. A method using ligation to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a plurality of capture beads each of having at least one transport probe affixed thereto;

preparing a plurality of reporter beads each having at least one signal probe affixed thereto;

mixing said capture beads, said reporter beads, and a sample to be tested for the presence of a target;

allowing any target present in the sample to bind to the transport and reporter probes thereby forming a dual bead complex including at least one reporter bead and one capture bead; and

performing a ligation reaction to introduce a covalent bond between the transport probes and the reporter probes to thereby strengthen the bond between the capture bead and the reporter bead so that when the dual bead complexes are processed, said strengthened bond withstands any external forces acting thereon.

9. The method according to claim 24 including the further steps of:

isolating the dual bead complex from solution to obtain an isolate;

exposing the isolate to a capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex to indicate that the target agent is present in the sample.

10. The method according to claim 24 wherein said mixing, allowing, and performing steps are carried out in a trackable optical bio-disc.

11. The method according to claim 25 wherein said isolating, exposing, and detecting steps are performed in a trackable optical bio-disc.

12. A method using a dual bead complex having a cleavable spacer to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing said dual bead complex including at least one reporter bead and at least one capture bead, said capture bead has at least one transport probe and said reporter bead has at least one signal probe, said beads being linked together by said cleavable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

cleaving said cleavable spacer to thereby dissociate any dual bead complex not associated with said target such that only the dual bead complex having target bound thereto remain in the dual bead formation;

performing a ligation reaction to introduce a covalent bond between said transport probe and said signal probe to thereby strengthen the bond between the capture bead and the reporter bead; and

detecting the presence of any intact dual bead complex.

13. A method using a dual bead complex having a displaceable spacer to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing said dual bead complex including at least one reporter bead and at least one capture bead, the beads being linked together by said displaceable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

displacing said displaceable spacer of said dual bead complex so that only complexes associated with the target remain in the dual bead formation;

performing a ligation reaction to introduce a covalent bond between the transport probe and the signal probe to thereby strengthen the bond between the capture bead and the reporter bead; and

detecting the presence and amount of any intact dual bead complex.

14. A method using a dual bead complex having a displaceable spacer to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing said dual bead complex including at least one reporter bead and at least one capture bead, the beads being linked together by said displaceable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

displacing said displaceable spacer of said dual bead complex using a displacement probe, so that only complexes associated with the target remain in the dual bead formation; and

detecting the presence and amount of any intact dual bead complex.